Pharmaceutical composition comprising citrulline

ABSTRACT

Embodiments of the present invention include compositions and pharmaceutical compositions comprising citrulline and Hmg-CoA reductase inhibitors. Further embodiments relate to the use of such composition treat subjects and stimulating nitric oxide synthase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.10/207,399, filed on Jul. 29, 2002, which is a continuation applicationof U.S. Ser. No. 09/293,392 filed Apr. 16, 1999, now U.S. Pat No.6,425,881, which is a continuation-in-part application of U.S. Ser. No.09/226,580 filed Jan. 7, 1999, now U.S. Pat. No. 6,239,172, which is acontinuation-in-part application of U.S. Ser. No. 09/833,842 filed Apr.10, 1997, now U.S. Pat. No. 5,968,983 which is a continuation-in-partapplication of U.S. Ser. No. 08/693,882 filed Aug. 5, 1996, now U.S.Pat. No. 5,767,160 dated Jun. 16, 1998, which is a continuation-in-partapplication of U.S. Ser. No. 08/321,051 filed Oct. 5, 1994, now U.S.Pat. No. 5,543,430 dated Aug. 6, 1996.

BACKGROUND OF THE INVENTION

Recently it has been established that a family of enzymes called NitricOxide Synthase (“NOS”) form nitric oxide from L-arginine, and the nitricoxide produced is responsible for the endothelium dependent relaxationand activation of soluble guanylate cyclase, neurotransmission in thecentral and peripheral nervous systems, and activated macrophagecytotoxicity.

Nitric Oxide Synthase, occurs in many distinct isoforms which include aconstitutive form (cNOS) and an inducible form (iNOS). The constitutiveform is present in normal endothelial cells, neurons and some othertissues. Formation of nitric oxide by the constitutive form inendothelial cells is thought to play an important role in normal bloodpressure regulation, prevention of endothelial dysfunction such ashyperlipodemia, arteriosclerosis, thrombosis, and restenosis. Theinducible form of nitric oxide synthase has been found to be present inactivated macrophages and is induced in vascular smooth muscle cells,for example, by various cytokines and/or microbial products.

The conversion of precursor substrates of EDNO such as L-arginine intonitric oxide is enzymatically catalyzed by NOS and the resultingby-product of the conversion of L-arginine is L-citrulline. Although itwas initially described in endothelium, NOS activity has now beendescribed in many cell types. Brain, endothelium, and macrophageisoforms appear to be products of a variety of genes that haveapproximately 50% amino acid identity. NOS in brain and in endotheliumhave very similar properties, the major differences being that brain NOSis cytosolic and the endothelial enzyme is mainly a membrane-associatedprotein.

Functionally, the constitutive form of Nitric Oxide Synthase (“cNOS”),which is the predominant synthase present in brain and endothelium, maybe active under basal conditions and can be further stimulated byincreases in intracellular calcium that occur in response toreceptor-mediated agonists or calcium ionophores. cNOS appears to be the“physiological” form of the enzyme and plays a role in a diverse groupof biologic processes. In vitro studies suggest that the activity ofnitric oxide synthase can be regulated in a negative feedback manner bynitric oxide itself.

In contrast to the cNOS, the inducible, calcium-independent form, iNOSwas initially only described in macrophages. It is now known thatinduction of nitric oxide synthase can occur in response to appropriatestimuli in many other cell types. This includes both cells that normallydo not express a constitutive form of nitric oxide synthase, such asvascular smooth muscle cells, as well as cells such as those of themyocardium that express considerable levels of the constitutive isoform.

iNOS exhibits negligible activity under basal conditions, but inresponse to factors such as lipopolysaccharide and certain cytokines,expression occurs over a period of hours. The induced form of the enzymeproduces much greater amounts of NO than the constitutive form, andinduced NOS appears to be the “pathophysiological” form of the enzymebecause high concentrations of NO produced by iNOS can be toxic tocells. Induction of iNOS can be inhibited-by glucocorticoids and somecytokines. Relatively little is known about postranscriptionalregulation of iNOS. Cytotoxic effects of NO are probably largelyindependent of guanylate cyclase and cyclic GMP formation.

It is known that administration of drugs consisting of nitric oxide, orreleasing nitric oxide, can inhibit restenosis after angioplasty.Chronic inhalation of nitric oxide inhibits restenosis followingballoon-induced vascular injury of the rat carotid artery. Oraladministration of NO donors (drugs which release nitric oxide) inhibitsrestenosis in rat and pig models of balloon angioplasty-induced vascularinjury.

The long term benefit of coronary balloon angioplasty and atherectomy islimited by the considerably high occurrence of symptomatic restenosis(40-50%) 3 to 6 months following the procedure. Restenosis is in partdue to myointimal hyperplasia, a process that narrows the vessel lumenand which is characterized by vascular smooth muscle cell migration andproliferation. Medical therapies to prevent restenosis have beenuniformly unsuccessful. Intravascular stents have been successfully usedto achieve optimal lumen gain, and to prevent significant remodeling.However, intimal thickening still plays a significant role in stentrestenosis.

The vascular architecture is maintained or remodeled in response to thechanges in the balance of paracrine factors. One of the substances thatparticipates in vascular homeostasis is endothelium derived nitric oxide(NO). NO is synthesized from the amino acid L-arginine by NO synthase.NO relaxes vascular smooth muscle and inhibits its proliferation. Inaddition, NO inhibits the interaction of circulating blood elements withthe vessel wall. NO activity is reduced in hypercholesterolemia andafter vascular injury. The administration of L-arginine alone has beenshown to restore vascular NO activity in animals and in humans withendothelial vasodilator dysfunction.

SUMMARY OF THE INVENTION

We have developed an approach to diminish the incidence of restenosisresulting from angioplasty and atherectomy, using an arginine basedmixture to enhance NO activity in the vessel wall.

The term “subject” as used herein means any mammal, including humans,where nitric oxide (“NO”) formation from arginine occurs. The methodsdescribed herein contemplate prophylactic use as well as curative use intherapy of an existing condition.

The term “native NO” as used herein refers to nitric oxide that isproduced through the bio-transformation of L-arginine or in theL-arginine dependent pathway. “EDRF” or “EDNO” may be usedinterchangeably with “native NO”. The term “endpoints” as used hereinrefers to clinical events encountered in the course of treatingcardiovascular disease, up to and including death (mortality).

“L-arginine” as used herein includes all biochemical equivalents (i.e.,salts, precursors, and its basic form). L-lysine may be considered abiological equivalent of L-arginine. Other bioequivalents of L-argininemay be arginase inhibitors, citrulline, ornithine, and hydralazine.

“To mix”, “mixing”, or “mixture(s)” as used herein means mixing asubstrate (ie., L-arginine) and another therapeutic agent agonist (e.g.,nitroglycerin or an Hmg-CoA reductase inhibitor): 1) prior toadministration (“in vitro mixing”); 2) mixing by simultaneous and/orconsecutive, but separate (e.g., separate intravenous lines)administration of substrate (L-arginine and agonist to cause “in vivomixing”; and 3) the administration of a NOS agonist after saturationwith a NOS substrate (e.g., L-arginine is administered to build up asupply in the body prior to administering the NOS agonist (nitroglycerinor Hmg-CoA reductase)); or any combination of the above which results inpre-determined amounts of a NOS agonist and a NOS substrate.

“Agonist” refers to an agent which stimulates the bio-transformation ofa NO precursor, such as L-arginine or L-lysine to EDNO or EDRF eitherthrough enzymatic activation, regulation or increasing gene expression(i.e., increased protein levels of c-NOS). Of course, either or both ofthese mechanisms may be acting simultaneously.

As used herein, the term “pharmaceutically acceptable carrier” refers toa carrier medium which does not interfere with the effectiveness of thebiological activity of the active ingredients and which is not toxic tothe hosts to which it is administered.

Methods and devices are provided for inhibiting the pathology associatedwith vascular injury, particularly during angioplasty and atherectomy. ANO producing mixture, preferably L-arginine and a NOS agonist, isintroduced either intraluminally or more preferably intramurally (forexample by a stent) to into the walls of the injured vessel in proximityto the injury in an amount to inhibit the pathology, e.g., restenosis,associated with the vascular injury. Various conventional deliverydevices may be used for the delivery of the therapeutic mixture.

The following examples or embodiments are offered by illustration, andnot by way of limitation.

The present invention is generally directed to treating vessels with amixture of L-arginine and an agent which enhances the biotransformationof L-arginine into NO. The incidents associated with restenosis areexpected to substantially reduced and prevented providing for a reducedincidence of restenosis.

In an embodiment of the present invention there is provided a method forreducing the probability of restenosis. The method comprises introducingintramurally or intraluminally to a site of an injury at apre-determined time from said injury to a second pre-determined time(e.g. not later than 6 hours thereafter), a therapeutic mixture, saidtherapeutic mixture including a biological equivalent of L-arginine; andan agent which enhances NO availability. In this embodiment it ispreferable that the agent stimulates conversion of L-arginine to NO bynitric oxide synthase, even more preferably the agent is a NOS agonist,even more preferably, the agent is a nitrate, and even more preferablythe agent is nitroglycerin. In this embodiment, it is preferable thatthe biological equivalent of L-arginine be selected from the groupconsisting of L-arginine and L-lysine. Alternatively, the biologicalequivalent of L-arginine is selected from the group consisting ofcitrulline and arginase inhibitors. The agent may also preventdegradation of NO. In this case it is preferable that the agent includeDOX.

In an alternative embodiment the present invention provides, a methodfor reducing the probability of restenosis resulting from vascularinjury, comprising: introducing intramurally and preferably proximallyto the site of said injury over a predetermined time (preferably about 2min to 0.5 h) an active agent, wherein the active agent includes anitric oxide precursor and an Hmg-CoA reductase inhibitor. In thisembodiment the nitric oxide precursor is preferably a biologicalequivalent of L-arginine, even more preferably a biological equivalentis selected from the group consisting of L-arginine or L-lysine or acombination of the two.

An alternative embodiment of the present invention provides a method forreducing the severity of restenosis, comprising introducing a biologicalequivalent of L-arginine intramurally and proximally to the site of saidinjury at a time from the time of said injury to a time not later than 6hours (in an aqueous solution at a concentration in the range of 20 to500 g/l); and introducing an agent which enhances the conversion of saidbiological equivalent of L-arginine into nitric oxide. It is preferablethat the method further includes the step of introducing an agent whichprevents the degradation of said nitric oxide. In this embodiment thestep introducing may be by means of a local delivery catheter.

In an alternative embodiment there is provided a method for reducing theprobability of-restenosis resulting from injury caused by angioplasty oratherectomy, comprising: introducing intramurally or intraluminally,preferably intramurally at the site of said injury a stent, said stenthaving a body comprised of L-arginine and a NOS agonist.

An alternative embodiment of the present invention provides a stenthaving a body comprising a NO precursor agent and a NOS agonist, the NOprecursor includes at least one of L-arginine or L-lysine, and the a NOprecursor agent and NOS agonist releasable under conditions present in ablood vessel.

An alternative embodiment of the present invention provides a stenthaving a body comprised of L-arginine and a nitrate, preferablynitroglycerin.

An alternative embodiment of the present invention provides a stenthaving a body comprised of L-arginine and an Hmg-CoA reductaseinhibitor, preferably atorvastatin or pravastatin.

An alternative embodiment of the present invention provides a stenthaving a body comprised of L-arginine and an angiogenic growth factor.

An alternative embodiment of the present invention provides a stenthaving a body comprised of L-arginine and DOX.

An alternative embodiment of the present invention provides ananti-restenosis device comprised of a body, said body including atherapeutic formulation. The therapeutic formulation of this embodimentincludes a NO precursor and a NO producing catalytic agent. In thisembodiment it is preferable that the NO precursor is L-arginine.Alternatively, the NO precursor may be L-lysine or a combination ofL-arginine and L-lysine. In this embodiment the NO precursor may be anarginase inhibitor. In this embodiment the NO producing catalytic agentis preferably a nitrate, preferably nitroglycerin. In this embodimentthe NO producing catalytic agent may also be an Hmg-CoA reductaseinhibitor, preferably statin, and more preferably, pravastatin. In thisembodiment the NO producing catalytic agent may be an angiogenic growthfactor. In this embodiment the NO producing catalytic agent may be DOX.

An alternative embodiment of the present invention provides a stentcomprised of a body, said body including an arginine based mixture, saidarginine based mixture including a biological equivalent of arginine andan agent which enhances the bioavailability of nitric oxide. In thisembodiment of the present invention, the biological equivalent ofarginine is L-arginine. Alternatively, the biological equivalent ofarginine may be L-lysine. In this embodiment the biological equivalentof arginine may be an arginase inhibitor. In this embodiment the agentwhich enhances the bioavailability of nitric oxide is preferably anitrate. In this embodiment the agent may be nitroglycerin. In thisembodiment the agent may be an Hmg-CoA reductase inhibitor. In thisembodiment the agent may a statin, preferably pravastatin. The mixturemay also include an angiogenic growth factor or DOX.

Finally as an alternative embodiment of the present invention, there isprovided a local in-dwelling intra-arterial eluting drug delivery devicecomprised of a body, said body incorporating a therapeutic mixturetherein, said therapeutic mixture including a NO precursor agent and anagent which enhances the conversion of the precursor agent to native NO.In this embodiment it is preferable that the NO precursor agent isL-arginine. In this embodiment the NO precursor may be L-lysine. In thisembodiment the NO precursor agent may be an arginase inhibitor. In thisembodiment the agent which enhances the conversion of the precursoragent to native NO may be a nitrate. In this embodiment the agent whichenhances the conversion of the precursor agent to native NO may benitroglycerin. In this embodiment the agent which enhances theconversion of the precursor agent to native NO may be an Hmg-CoAreductase inhibitor. In this embodiment the agent which enhances theconversion of the precursor agent to native NO may be a statin. In thisembodiment the agent which enhances the conversion of the precursoragent to native NO may be pravastatin. In this embodiment the agentwhich enhances the conversion of the precursor agent to native NO may bean antiogenic growth factor. In this embodiment the agent which enhancesthe conversion of the precursor agent to native NO maybe DOX.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is the top portion of a schematic representation of proposedL-arginine dependent and independent pathways.

FIG. 1B is the bottom portion flowing from FIG. 1A of a schematicrepresentation of the proposed L-arginine dependent and independentpathways.

FIG. 2 is a bar graph illustrating the NOS stimulating effect ofcombined administration of L-arginine and nitroglycerin on rat aorta.

FIG. 3 is a schematic representation of the proposed NOS activationpathway involving pravastatin.

FIG. 4 is a bar graph illustrating the stimulation of NOS withpravastatin.

FIG. 5 is a schematic representation of the proposed pathway involvingVegF.

FIG. 6 is a fragmentary view, partially in section, of a drug deliveryapparatus for use in the subject invention positioned in a blood vesselwith the dilatation balloon in its inflated state and containing atherapeutic mixture of the-present invention.

FIG. 7 is a fragmentary view, partially in section, of the drug deliveryapparatus positioned in a blood vessel and embodying iontophoresis meansto transport the drug across the balloon surface.

FIG. 8 is a perspective view of a catheter loaded with a stent in acoronary artery narrowed by a lesion.

FIG. 9 is a perspective view of an expanded stent holding the lumen ofthe coronary artery open.

FIG. 10 is a schematic representation of the dynamics of L-argininesupply to NOS.

FIG. 11 indicates the effect of B^(α+) and y⁺ transporters on cellularuptake of [³H]-L-arginine.

FIG. 12 indicates the effect of bradykinin (BK, 1αM) on y⁺ transport of[³H]-LA in bovine aortic endothelial cells.

FIG. 13 indicates the effect of substance P (SP, 1 μM) on y⁺ transportof [³H]-LA in bovine aortic endothelial cells.

FIG. 14 indicates the effect of acetylcholine (Ach, 5 μM) on y⁺transport of [³H]-LA in bovine aortic endothelial cells.

FIG. 15 indicates the effect of s-nitroso-acetyl-penicillamin (SNAP, 200μM; equivalent to 0.4 μM NO) on y⁺ transport of [³H]-LA in bovine aorticendothelial cells.

FIG. 16 indicates the effect of dipropylenetriamine NONOate (DPTA,10-0.01 μM; equivalent to 20-0.02 μM NO) on y⁺ transport of [³H]-LA inbovine aortic endothelial cells.

FIG. 17 indicates the effect of L-arginine (LA, 5×10⁻⁴M) andn-ω-nitro-L-arginine methyl ester (L-NAME, 5×10⁻⁴M) on substance P (SP,1 μM) or calcium ionaphore, A-23187 (CI, 1 μM) induced superoxide anion(O₂*—) formation in bovine aortic endothelial cells (BAEC).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Devices and methods are provided for the treatment of pathologiesassociated with vascular injury, particularly in relation to angioplastyand atherectomy. Of particular interest is the injury referred to asrestenosis, which results from the migration and proliferation ofvascular smooth muscle cells into the intima of the vessel as well asaccretions associated with the atherosclerosis.

The method provides introducing to or into the vessel walls at the siteof injury a therapeutic mixture which includes at least a NO precursorand more preferably a combination of a NO precursor and an agent whichenhances the conversion of the NO precursor to NO and which results inthe enhancement of NO production in the cells at the site of injury.Various delivery systems may be employed which result in the therapeuticmixture infusing into the vessel wall, and being available to the cellsfor NO production. Devices which may be employed include drug deliveryballoons, e.g., porous, sonophoretic, and iontophoretic balloons, asexemplified by the devices depicted in WO92/11895, WO95/05866 andWO96/08286, which are incorporated herein by reference thereto. Also,stents may be employed where the stent carries the therapeutic mixtureincluding the NO precursor agent. Preferably, the stent is convenientlyintroduced with a catheter, so that both short and long term delivery ofthe NO precursor agent can be provided for enhanced protection againstblockage.

Using catheters for the delivery of the therapeutic mixture will beconsidered first. The NO precursor based mixture is introduced in adelivery balloon for transport by a catheter to the site of injury. Theballoon may then be expanded under pressure driving the therapeuticmixture from the balloon into or near the surrounding vessel wall. Theamount of mixture which is employed may vary depending upon the natureof the mixture, the region to be treated, and the loss of the mixturefrom the region. The infusion of the mixture is maintained forsufficient time to ensure that the cells and extracellular matrix in theinjury region are exposed to the mixture, so as to enhance theproduction of NO by these cells.

The mixture is preferably a combination of active ingredients. Ofparticular interest are the amino acids, L-arginine and L-lysine,individually or in combination, as a mixture or as an oligopeptide, or abiologically equivalent compound, such as low molecular weightoligopeptides, having from about 2-10, usually 2-6 amino acids, oracetylated amino acids and oligopeptides, etc. in combination with anagent which enhances the conversion of the NO precursor.

A physiologically acceptable medium will be employed, normally anaqueous medium, which may be deionized water, saline, phosphate bufferedsaline, etc. The amount of the active NO precursor agent will varydepending upon the particular agent employed, the other additivespresent, etc. Generally, as exemplified by L-arginine, at least about 50mg will be present, and not more than about 5 g, usually at least about100 mg, and not more than about 2 g, frequently at least about 500 mg.The concentration may be varied widely, generally ranging from about20-500, more usually from about 50-250 g/l.

The subject methodology is employed with hosts who have sufferedvascular injury, as caused by angioplasty and atherectomies. The timefor the administration of the therapeutic mixture may be varied widely,providing a single administration or multiple administrations over arelatively short time period in relation to the time of injury.Generally, treatment may be before, concurrently or after the injury,usually within 2 weeks of the injury, if before, and not more than about8 weeks, usually not more than about 6 weeks, preferably in the range of0-6 weeks (where 0 intends concurrently or shortly after the priorprocedure, within 6 hours).

It is expected that with one treatment of the NO precursor agent at orabout the time of the injury, before or shortly thereafter, one willobserve enhanced vascular NO production and reduced intimal thickening,so as to substantially reduce the potential for restenosis.

In conjunction with the intraluminal or intramural deliver of thetherapeutic mixture by the catheter, a stent may be introduced at thesite of vascular injury. The stent may be biodegradable ornon-biodegradable, may be prepared from various materials, such asmetals, ceramics, plastics or combinations thereof. Biodegradableplastics, such as polyesters of hydroxycarboxylic acids, are ofparticular interest. Numerous stents have been reported in theliterature and have found commercial acceptance. An example of the typeof stent which may be modified to deliver an arginine based mixture(including an agent which enhances production of NO) is shown in U.S.Pat. Nos. 5,665,077, 5,482,925, and 5,405,919, each of which areincorporated by reference hereto in their entirety.

Depending on the nature of the stent, the stent may have the therapeuticmixture incorporated in the body of the stent or coated thereon. Forincorporation, normally a biodegradable plastic stent will be used whichwill release the therapeutic mixture while supporting the vessel andprotecting against restenosis. In the fabrication of the stent, thebiodegradable matrix may be formed by any convenient means known in theart. Alternatively, the stent may be coated with the therapeuticmixture, using an adhesive or coating which will allow for controlledrelease of the therapeutic mixture. The stent may also be comprised ofthe NO precursor agent with simultaneous or consecutive administrationof the other active agent (e.g., a NOS agonist such as nitroglycerin oran Hmg-CoA reductase inhibitor such as pravastatin). The stent may bedipped, sprayed or otherwise coated with a composition containing the NOprecursor agent or the therapeutic mixture and a matrix, such as thebiodegradable polymers described above, a physiologically acceptableadhesive, proteins, polysaccharides or the like. By appropriate choiceof the material for the stent and/or the coating comprising the NOprecursor agent or therapeutic mixture, a physiologically active amountof the NO precursor agent and/or therapeutic mixture may be maintainedat the site of the vascular injury, usually at least one day and up to aweek or more.

The amount of the NO precursor agent or therapeutic mixture will bedetermined empirically in accordance with known techniques using animalmodels. The amount of the NO precursor agent (e.g., L-arginine) employedshould provide a physiologically effective amount to reduceproliferation of vascular smooth muscle cells and maintain the dilationof the vessel, while preventing restenosis.

FIG. 1A and FIG. 1B illustrate a schematic representation of theproposed mechanism of action elicited by nitrovasodilators on both agenerator cell and a target cell and their interrelationship. It appearsthat nitroglycerin or glycerol trinitrate's (GTN) mechanism of action isboth L-arginine dependent and L-arginine independent and thisimplication has far reaching effects regarding the development andtreatment of nitroglycerin tolerance and reducing clinical endpoints andmortality. Research into the area of cNOS activation reveals a number ofagonist of cNOS some of which have been described in U.S. Pat. No.5,543,430 and U.S. Pat. No. 5,767,160, both of which are herebyincorporated by reference in its entirety. The following discussion willfocus on smooth muscle and myocyte relaxation stimulated bynitrovasodilators wherein the nitric oxide synthase is cNOS, theconstitutive form of nitric oxide synthase, the generator cells areendothelial cells and the target cells are vascular smooth muscle cells.This illustration is not intended to imply any cellular relationshipbetween the various sites of action, but rather meant to illustratetheir functional relationship.

As shown in FIGS. 1A and 1B the production of NO may result from avariety of sources and mechanisms which are discussed in detail inIgnarro, (Louis J. PhD., 1991, Pharmacology of Endothelium-DerivedNitric Oxide and Nitrovasodilators, The Western Journal of Medicine, pp.51-62.). In the L-arginine independent or non-endothelium dependentpathway the activation of Guanylate Cyclase (GC) by Nitric Oxide (NO)depends on the type of nitrovasodilator used. Inorganic Nitrite (NO₂—)is charged and only limited amounts can permeate the cell, butintracellular nitrite can be converted to NO. Lipophilic organic nitrateesters (R—OH) are converted into NO by acidic thiol (R—SH) facilitatedreactions. S-Nitrosothiols (R—SNO) are labile intermediates thatdecompose spontaneously and produce NO. It is thought that one of themechanisms by which thiols potentiate the action of nitroglycerin andreverse to some degree tolerance to nitroglycerin is through the directreaction between the thiol (R—SH) and nitroglycerin (GTN) to form thelabile intermediate S-Nitrosothiol (R—SNO), which decompose as describedabove (R—SH+GTN→R—SNO is not shown). A nonenzymatic formation ofexogenous NO is thought to occur with thiol sources such as cysteine,dithiothreitol, N-acetylcysteine, mercaptosuccinic acid, thiosalicylicacid, and methylthiosalicylic acid.

It is hypothesized that the tolerance to nitroglycerin may involve asecondary pathway, or indeed, this “secondary pathway” may be theprimary pathway. This “secondary pathway” is the L-arginine dependentpathway or endothelium dependent pathway shown in FIGS. 1A and 1B. Asseen in FIG. 1A, the generator cell is known to have several receptormediated agonists such as Endothelium B receptor (ETB); acetylcholine(Ach); substance P (SP), Histamine (H); arginine vasopressin (AVP);bradykinin (BK); Adenosine Triphosphate (ATP); Prostaglandin F_(2α)(F_(2α)); Oxytocin, (OT); and the calcium ionophore (A23187) whichstimulate the production of NOS. However, until now it has not beenspeculated that nitroglycerin may serve the dual role of agonist forNOS, and pro-drug for the sulfhydryl mediated L-arginine independentpathway.

It has been discovered that combining L-arginine or biologicallyequivalents thereto with nitroglycerin prior to administration overcomesthe resistance or tolerance level normally established whenadministering nitroglycerin alone. It is believed that NOS may bestimulated by nitroglycerin and that premixing with L-arginine has abeneficial effect that may be due to a complex or coordinate formationbetween nitroglycerin and L-arginine. Excess L-arginine providesadditional substrate for the stimulated nitric oxide synthase whichcatalyzes the biotransformation of L-arginine into nitric oxide. As usedherein a “biological equivalent” is an agent or composition, orcombination thereof, which has a similar biological function or effectas the agent or composition to which it is being deemed equivalent. Forexample, a biological equivalent of arginine is a chemical compound orcombination of chemical compounds which has the same or similarbiological function or effect as arginine. Lysine may be considered abiological equivalent of arginine. Other expected biological equivalentsinclude citrulline, arginase inhibitors, hydralazine, and ornitine.Previously it was thought that nitroglycerin had no effect on thebiotransformation of L-arginine into “native” nitric oxide, but it isnow believed that nitroglycerin or possibly a nitroglycerin complex orcoordinate with L-arginine has a stimulating effect on NOS.

Combining L-arginine and nitroglycerin may also result in a combinedarterial and venous dilatory effect. Used alone nitroglycerin isprincipally a venodilator at low doses although it can become aveno-arterial dilator at high doses and causes rapid increase in heartbeat due to its venous pooling, while L-arginine on the other hand whenused alone is principally an arterial dilator. Therefore, combining thetwo results in balanced arterial and venodilatory effect which counterbalances the tendencies of one or the other to produce tachycardia whichis adverse to ischemia in an evolving myocardial infarction.

Another mechanism of benefit from the combination relates to the factthat used alone nitroglycerin is of only minimal benefit in limitingreperfusion injury with patients who have had recent heart attacks andabrupt restoration of blood flow. The same thing is seen in patients whoare undergoing re-establishment of blood flow after coronary bypassoperations coming off the bypass pump.

We discovered that dogs treated to a floor of nitroglycerin effect couldbe made further responsive by the co-administration of nitroglycerin andL-arginine in water in a manner similar to that commonly seen clinicallywith the addition of sodium nitroprusside (SNP) to nitroglycerin;however, when compared to SNP, L-arginine combined with nitroglycerinhad much more favorable hemodynamic effects. Compared to SNP, vascularresistance was reduced by 50%, cardiac output doubled, and contractilityincreased. This led to the hypothesis that the combination of L-arginineand nitroglycerine was generating EDRF as opposed to SNP which is knownto produce nitric oxide in a direct fashion. The following keycorresponds to the bar graph shown in FIG. 2.

A. Control—Basal. This represents cGMP activity at baseline that wasgenerated by resting NO sources of soluble guanylate cyclase activation,ie., baseline.

B. L-arginine Group. This represents cGMP activity generated byL-arginine and EDRF (endogenous or “native” NO production).

C. Nitroglycerin Group. (L-arginine plus nitroglycerin) The cGMPactivity represents the sum of B (L-arginine) plus nitroglycerininduction of CNOS and the subsequent EDRF produced in addition to nitricoxide from nitroglycerin by the L-arginine independent pathway (pro-drugeffects).

D. L-NAME Group. L-arginine (L-arginine plus nitroglycerin plus L-NAME).Represents cGMP activity from nitroglycerin enzymatic conversion alonesince L-NAME used in excess inhibits NOS derived EDRF from all sources.

E. L-arginine+L-NAME—represents cGMP activity due to non-nitric oxidesources activating soluble guanylate cyclase activation and wassubtracted from all measurements to eliminate effects of non NOactivation of cGMP.

From this it is apparent that: Total NO from nitroglycerin is C-B; NOfrom enzymatic degradation of nitroglycerin to NO equals D-E; EDRF (NOS)stimulation from nitroglycerin=(C-B)-(D-E)

FIG. 2 summarizes our results with a bar graph representative of therespective detected picomols of cGMP/100 mg wet tissue. Although notshown in FIG. 2, when nitroglycerin and L-NAME were combined in theabsence of L-arginine, similar results were obtained regarding cGMPproduction. In both FIG. 2 the bar labeled NOS is the amount of “native”NO produced which is total NO minus the NO produced via the L-arginineindependent pathway.

Nitroglycerin resistance—tolerance has frustrated cardiologists andpharmacologists since 1888. (Stewart D. D., 1888, Remarkable Toleranceto Nitroglycerin. Philadelphia Polyclinic. 172-5.) Our results supportthe hypothesis outlined in FIG. 1B and clarify the mechanism ofnitroglycerin tolerance. It is believed that an additional nitroglycerinactivation site is CNOS in the endothelial cell. Under conditionsleading to tolerance the agonist effect of nitroglycerin on cNOSinduction leads to a depletion of L-arginine in the endothelial cellsecondary to rate limitations in active L-arginine transport pumpkinetics in FIG. 1A and FIG. 1B. This creates a supply demand mismatchsituation at the membrane uptake step and explains why arginine is ratelimiting. This may also explain why during administration ofnitroglycerin a nitrate free interval is required. It is believed thatthis is necessary so that the endothelial cells can replete thedeficient L-arginine by active transport. By adding L-arginine whenadministering nitroglycerin it is believed that EDRF can be generated,and in the process a significant reduction in clinical and mortalityendpoints can be obtained relative to using nitroglycerin alone or incombination with SNP or other donors of exogenous NO.

It has been shown that nitroglycerin applied at the site of intimalinjury following balloon angioplasty reduces the formation of medicalcellular proliferation. However, intimal and neointimal proliferationwere not reduced. This was thought to be secondary to the development oftolerance to nitroglycerin. We have shown that tolerance tonitroglycerin may in fact be related to its function as a NOS agonist.The activation of Nitric Oxide Synthase which results in a developmentof tolerance to the effectiveness of the nitroglycerin and the fact thattolerance to nitroglycerin can be overcome by the concomitantadministration locally of L-Arginine, its salts or of its biologicalequivalents, such as Lysine provide a heretofore unexpected benefit ofthe application of a mixture of nitroglycerin and L-arginineutilized anacute or chronic intrarterial site-specific locally indwelling and/orelating anterestenosis drug delivery device at the site of ballooninjury.

In another embodiment of the invention, therapeutically effectiveamounts of L-arginine and inhibitors of Hmg-CoA reductase are mixed at aphysiologically acceptable pH and administered to a patient.

It appears that inhibitors of Hmg-CoA reductase may have dualapplicability in the treatment of hypertension and cardiovasculardiseases such that they act as both an inhibitor of the intrinsicbiosynthesis of cholesterol and a stimulator or agonist of nitric oxidesynthase. The fact that Hmg-CoA reductase may be agonist or stimulant ofnitric oxide synthase has remarkable implications. Mixing inhibitors ofHmg-CoA reductase “in vitro” or “in vivo” with L-arginine has been foundto have an unforeseen beneficial effect that is most likely due toexcess L-arginine providing additional substrate for the nitric oxidesynthase and the NOS being catalyzed to enzymatically increase thebio-transformation of L-arginine into nitric oxide.

L-arginine may be used in conjunction with virtually any of the familyof those substances known as Hmg-CoA reductase inhibitors. These aretaught for example in U.S. Pat. Nos. 4,857,522, 5,190,970, and6,461,039, all of which are hereby incorporated by reference for thisteaching. Those particular Hmg-CoA reductase inhibitors most preferredfor use in conjunction with the present formulation as selected from thegroup consisting of: atorvastatin, cerivastatin, simvastatin,lovastatin, pravastatin, compactin, fluvastatin, and dalvastatin. U.S.Pat. No. 5,316,765 cites a number of these Hmg-CoA reductase inhibitorsand is hereby incorporated by reference in its entirety. In particularlypreferred embodiments of the present invention, the Hmg-CoA reductaseinhibitor utilized is pravastatin or atorvastatin. In an even moreparticularly preferred embodiments, the administration of the presentinvention includes the Hmg-CoA reductase inhibitor pravastatin.

As part of a “mixture”, the Hmg-CoA reductase inhibitor is includedtogether with L-arginine and clinically effective weight ratios ofbetween 1:2 to 1:150. Even more particularly, the ratio of the Hmg-CoAreductase L-arginine in the formulation is between 1:5 to 1:100. Themost preferred embodiment of the “mixture” the ratio of Hmg-CoAreductase inhibitor, most particularly pravastatin, to L-arginine is1:50. The range of ratios of an Hmg-CoA reductase inhibitor toL-arginine may be employed with virtually any Hmg-CoA reductaseinhibitor.

Where the particular Hmg-CoA reductase inhibitor is pravastatin, theratio of pravastatin to L-arginine is preferably within the range 1:2 to1:50, Wt/Wt. For example, pravastatin/L-arginine at a ratio of 1:2 wouldinclude 40 mg/day pravastatin with 80 mg/day L-arginine. Where the ratioof pravastatin/L-arginine is at a ratio of 1:20, for example, 20 mg/daypravastatin would be administered with 400 mg/day L-arginine. Weightratio of ingredients described herein in regard to the Hmg-CoA reductaseinhibitors, lovastatin, pravastatin and atorvastatin are applicable forany Hmg-CoA reductase inhibitor. The amounts above have been found to beeffective, however, each route of administration (e.g., IV, oral,tranadermal, etc.) will vary in their requirements.

Even more particularly, the presently disclosed “mixtures” may bedescribed in terms of their relative concentrations (grams) administeredas part of a continuous daily and/or monthly regimen. In one particularembodiment, the formulation is administered so as to provide the patientwith between 20-40 milligrams per day of the Hmg-CoA reductase inhibitor(e.g., pravastatin) together with a daily dose of L-arginine of between100 to 200 mg per day. Most preferably, the Hmg-CoA reductase inhibitor,such as lovastatin, is administered at a daily dose of about 20 mg perday together with a dose of about 200 mg per day L-arginine. Thisparticular embodiment of the claimed formulation should maintain withinthe patient efficient levels of the formulation.

The Hmg-CoA reductase inhibitors of the present invention are alsocharacterized by an ability to stimulate receptor-mediated clearance ofhepatic low-density lipoproteins (LDL), as an anti-hypercholesterolemic,and as a competitive inhibitor of Hmg-CoA reductase.

The preparation of lovastatin, simvastatin, and pravastatin have beendescribed in the patent literature. The preparation of XU-62-320(fluvastatin) is described in WIPO Patent W084/02131. BMY 22089(13), CI981(14), HR 780(15), and SQ 33,600 are also described in the literaturecited, and are specifically incorporated herein by reference for thepurpose of even more fully describing the chemical structure andsynthesis of these Hmg-CoA reductase inhibitors. These methods ofpreparation are hereby incorporated by reference in their entirety.

Also within the scope of those Hmg-CoA reductase inhibitors of thepresent invention are included the bio-active metabolites of thoseHmg-CoA reductase inhibitors described here, such as pravastatin sodium(the bio-active metabolite of mevastatin).

Any one or several of the Hmg-CoA reductase inhibitor compounds may bemixed with L-arginine or substrate precursor to endogenous nitric oxideto provide a therapeutically effective mixture. This therapeuticallyeffective mixture can then be incorporated into a stent or otherdelivery device.

Until our discovery there was no link between the bio-transformation ofL-arginine into “native” nitric oxide and anti-hypocholesterolemicHmg-CoA reductase inhibitors. However, it is now believed that Hmg-CoAreductase inhibitors may have an affect on NOS. It appears the mixtureof inhibitors of Hmg-CoA reductase and biological equivalents L-argininemay have a heretofore unexpected affect on cNOS stimulation.Administering the two also provides adequate substrate for NOSprocessing of L-arginine since the L-arginine is added in excess whileat the same time stimulation the enzymatic activity of NOS. Whether itis a synergistic effect or additive effect, what is clear is that“mixing” a precursor substrate of “native” nitric oxide with a Hmg-CoAreductase inhibitor results in a heretofore unexpected increase in NOproduction.

To demonstrate this, the direct effects of acteylcholine and pravastatinon NO production in bovine aortic endothelial cells (BAEC) wasdetermined using a highly sensitive photometric assay for conversion ofoxyhemoglobin to methemoglobin. NO oxidize; oxyhemoglobin (HbO₂) tomethemoglobin (metHb) in the following reaction HbO₂+NO−metHb+NO₃. Theamount of NO produced by endothelial cells was quantified by measuringthe change in absorbance as HbO₂ oxidizes to metHb. Oxyhemoglobin has aabsorbance peak at 415 nm, while methb has a 406 nm absorbance peak. Bysubtracting the absorbance of metHb from HbO₂, the concentration of NOcan be assessed. The general method was patterned after that of Feelischet al., (Biochem. and Biophy. Res. Comm. 1991; 180, Nc I:286-293).

FIG. 4 is a bar graph of the data generated which illustrates theeffects of acetylcholine and pravastatin (10⁻⁶ and 10⁻⁵ M) administeredfor 3 min periods into the cell/bead perfusion system on NO productionwith: 1) 10⁻⁵ M L-arginine in control (basic) buffer, 2) 10⁻³ M ofL-NAME in buffer, and 3) 10⁻³ M of L-arginine in buffer. Responses aretransient elevations in NO production above basal levels. Data forresponses in L-NAME and L-arginine augmented buffer are presented aspercent of response in control buffer (100%); numbers in basic bufferbars indicate absolute production of NO in nmole *min. The remaining twobars denote differences between responses in L-NAME buffer vs both basicand L-arginine added buffers.

The effects of pravastatin on activity of endothelial cells in producingNO were compared with those of actetylcholine, which is known tospecifically stimulate NO production by NOS activity. Addingacetylcholine to the buffer superfusion bovine aortic endothelial cells(BAECs) grown on beads increased their production of NO as measured byoxidation of oxyhemoglobin to methemoglobin. Acetylcholine produced atransient, concentration-related increase in NO above baseline levels.In basic buffer containing 5×10⁻⁵M L-arginine, and there wasapproximately a two fold increase in NO production between 10⁻⁵ ML-arginine, there was approximately a two fold increase in NO productionbetween 10⁻⁵ and 10⁻⁶ M acetylcholine. Subsequent treatment of thesecells with buffer containing L-NAME, 10⁻³ M markedly reducedacetylcholine-induced production of NO by 80%. When this L-NAME bufferwas replaced with another containing increased L-arginine (10⁻³ M),acetylcholine-elicited production of NO returned to control levels.

Pravastatin also caused a concentration-related increase in NOproduction above baseline levels. There was a larger increment inresponse to the 10⁻⁵ M concentrations of pravastatin (˜3 X) comparedwith that of acetylcholine. Superfusion of the cell suspension withL-NAME (10⁻³ M), also blunted NO production in response to pravastatin.This suggests that NO production is due at least in part to NOSactivity. Subsequent perfusion of the cells with a buffer containingL-arginine 10⁻³ M resulted in a return in NO production to a level abovethe amount induced by the Pravastatin in control (basis) buffer. Thisrestoration of response to Pravastatin after L-arginine addition wasgreater than that observed for acetylcholine. Administration ofPravastatin or acetylcholine into a perfusion system containing onlybeads without cells did not induce metHb/NO production.

In an alternative embodiment of the present invention, therapeuticallyeffective amounts of L-arginine and therapeutically effective amounts ofa macrophage secretory product or angiogenic growth factor are mixed ata physiologically acceptable pH and delivered for example by a stent.

Many of the NOS agonists originally identified have also been implicatedin angiogenesis. Substance P (“SP”), a secretory product, is identifiedherein as a cNOS agonist. Other secretory products (e.g., thoseidentified in “Macrophages and angiogenesis” by Sunderkotter et al. (JLeukoc Biol 1994 Mar; 55(3):410-22)) may also be expected to be agonistsof NOS. Bradykinin (“BK”), a NOS agonist, has also been implicated as apossible angiogenic factor. Angiogenic growth factors like thoseidentified in Table I stimulate the growth of new blood vessels (e.g.,in vascular beds such as the coronary, peripheral, etc.) previouslyoccluded with atherosclerotic obstructions. Angiogenic growth factorsare proteins which were initially discovered as agents responsible forthe growth of new blood vessels which maintain the growth and spread ofcancerous tumors (neovascularization). Two of the angiogenic growthfactors, vascular endothelial growth factor (VEGF) and basicfibroblastic growth factor (bFGF), have been infused into catheters,used at the time of generating coronary and peripheral arteriograms, andhave resulted in the growth of significant new collateral blood vesselsin the region of ischemia producing vascular atherosclerotic occlusions.In this way, the symptoms of ischemia are lessened. The term applied tothis treatment approach is “therapeutic angiogenesis.”

Like angiogenic agents Substance P and Bradykinin, VEGF and bFGF alsoappear to act as NOS agonists, specifically cNOS. It appears theresultant EDNO produced is in large part responsible for the newcollateral vessel growth (“collaterar”) which in turn is responsible forthe improvement in symptoms of ischemia seen in therapeuticangiogenesis. Furthermore, it has also been shown that the collateralresponses to both VEGF and bFGF can be magnified significantly withL-arginine supplementation. Therefore, angiogenic growth factors,preferably VEGF and bFGF, appear to have dual applicability in thetreatment of hypertension and cardiovascular diseases in that they bothstimulate therapeutic angiogenesis and activity of Nitric OxideSynthase. It also appears that the overall therapeutic angiogenic resultwith angiogenic growth factors is augmented to the extent they act asagonists of NOS. The fact that angiogenic growth factors are agonists orstimulators of nitric oxide synthase has important implications. Mixingangiogenic growth factors “in vitro” or “in vivo” with L-arginine mayhave an unforeseen beneficial effect that is associated with excessL-arginine providing additional substrate for NOS and the NOS beingcatalyzed to enzymatically increase the bio-transformation of L-arginineinto nitric oxide (EDRF or EDNO) which would in turn amplify the overalltherapeutic effect.

Stimulation of NOS by angiogenic growth factor(s) in the presence ofexcess L-arginine or other substrate precursor of native NO may be usedto prevent, treat, arrest, or ameliorate any disease or condition whichis positively affected by NO production. Such conditions includehypertensive cardiocerebrorenovascular diseases and their symptoms aswell as non-hypertensive cardiocerebrorenovascular diseases. The mixtureis particularly useful for subjects in need of native NO production fortherapeutic angiogenesis. Thus the application for intraluminal orintramural administration (such as by stent—either eluting Stents orcoated stents.

In this embodiment, L-arginine is used in conjunction with any of thefamily of those substances known as angiogenic growth factors. However,those particular angiogenic growth factors most preferred for use inconjunction with the present formulation are selected from the groupconsisting of VEGF and bFGF and even more preferably VEGF. Any of theagonists of Table I may be suitable candidates for use in combinationwith L-arginine.

As part of a “mixture”, the angiogenic growth factor is includedtogether with L-arginine and clinically effective weight ratios ofbetween 1:2 to 1:150. Even more particularly, the ratio of theangiogenic growth factor to L-arginine in the formulation is between 1:5to 1:100. The most preferred embodiment of the “mixture” the ratio ofangiogenic growth factor, more preferably VEGF or bFGF, to L-arginine is1:50. VEGF can be obtained from Genentech (South San Francisco, Calif.)and bFGF can be obtained from R&D Systems (Minneapolis, Minn.). Therange of ratios of an angiogenic growth factor to L-arginine may beemployed with virtually any of the angiogenic growth factors.

Where the particular angiogenic growth factor is VEGF the ratio of VEGFto L-arginine is preferably within the range 1:2 to 1:50, Wt/Wt. Forexample, VEGF/L-arginine at a ratio of 1:2 would include 40 mg/day VEGFwith 80 mg/day L-arginine. Where the ratio of VEGF/L-arginine is at aratio of 1:20, for example, 20 mg/day VEGF would be administered with400 mg/day L-arginine. Weight ratio of ingredients described herein inregard to VEGF or bFGF are generally applicable. The amounts above havebeen found to be effective, however, each route of administration (ie.,IV, oral, transdermal, intracoronary, intra-arterial, etc.) may vary intheir requirements.

FIG. 5 is a schematic illustration of a proposed mechanism of action ofpreferred substances (e.g., angiogenic growth factors) and arginine andis not intended to imply any cellular relationship or geography of thevarious sites of action, but rather meant to illustrate their functionalrelationship. FIG. 5 lists certain preferred agents as angiogenic agentsand is meant as a representative sampling. SP represents Substance P andGF representing select Growth Factors.

As indicated, various delivery devices may be employed for the deliveryof the active agent(s). FIG. 6 illustrates the drug delivery apparatuswith the balloon 12 in its inflated state and within an arterial vesselin which the vessel walls are indicated by the reference numeral 15.During percutaneous transluminal coronary angioplasty (“PCTA”)procedures, the guide wire 10 is first inserted into the selected arteryto a point past the stenotic lesion. The dilatation catheter includingthe catheter body 11 and the balloon 12 is then advanced along the guidewire 10 to the desired position in the arterial system in which theballoon portion 12 traverses or crosses the stenotic lesion. The balloon12 is then inflated by introducing the solution containing thetherapeutic mixture (or the NO precursor if subsequent or simultaneousdelivery of a second agent is being employed) through the balloon lumen14 into the interior chamber 13 of the balloon 12. During inflation, theouter surfaces of the balloon 12 press outwardly against the innersurfaces of the vessel wall 15 to expand or dilate the vessel in thearea of the stenotic lesion, thus performing the angioplasty portion ofthe method as well as the intramural introduction of the therapeuticagent(s) into the vessel wall.

The porous balloon may be made from any of the conventional materialsused for this purpose. These include cellulose acetate, polyvinylchloride, polysulfone, polyacrylonitrile, polyurethanes, natural andsynthetic elastomers, polyolefins, polyesters, fluoropolymers, etc.Usually the film thickness will be in the range of about 10 Å to 1μ,with a nominal pore size of about 0.05 to 1μ Alternatively, a local drugdelivery system may be employed where the agent(s) is delivered to thevessel wall by channels that are on the exterior surface of the balloon.The balloon is placed into the diseased vessel segment as describedabove. The balloon is then inflated in the usual manner (using saline,usually containing a contrast agent), placing the channels (on thesurface of the balloon) in contact with the vessel wall. The therapeuticsolution is then infused under pressure into the channels. Perforationsin the channels allow the solution to exit and jet into the vessel wallunder pressure to enhance intramural delivery.

Alternatively, a local drug delivery system may be employed where theagent(s) is delivered to the vessel wall by channels that are on theexterior surface of the balloon. The balloon is placed into the diseasedvessel segment as described above. The balloon is then inflated in theusual manner (using saline, usually containing a contrast agent),placing the channels (on the surface of the balloon) in contact with thevessel wall. The therapeutic solution is then infused under pressureinto the channels. Perforations in the channels allow the solution toexit and jet into the vessel wall under pressure to enhance intramuraldelivery.

Alternatively, an iontophoretic approach may be used. FIG. 7 illustratesa structure utilizing iontophoresis to assist in driving the activeagent(s) across the balloon wall 26 and into contact with the vesselwalls 15. One electrode 28, the catheter electrode, is located on orwithin the catheter body 11, while the other electrode 31, the bodysurface electrode, is located on the body surface or within the body ofthe patient. An electrical current for the iontophoretic process isproduced between the electrodes 28 and 31 by an external power source 30through the electrical leads 29 and 33, respectively. Direct current maybe used, although other wave forms are also utilized (e.g., a series ofrectangular waves producing a frequency of 100 Hz or greater).

During operation of the iontophoretic device, the balloon 26 is firstpositioned across the stenotic lesion. The balloon interior 27 is theninflated with the drug in the lumen 23. As the balloon expands, itcauses the artery to dilate. This is followed by activating the powersupply 30, thereby creating a current between the electrode 28 and theelectrode 31 which passes through the balloon wall 26. This currentdrives or drags the agent(s) (e.g., NO precursor and nitroglycerin)within the chamber 27 across the wall and into contact with thesurrounding vessel wall 15 and vascular tissue.

In FIG. 8 is shown a device 30 comprising a catheter 32 carrying a meshstent 34 encircling balloon 36 in its collapsed state. The mesh stent 34would be covered for example with a slow release layer of L-arginine/NOSagonist or L-arginine/statin (e.g., pravastatin) containingpoly(glycolide-lactide) 38. The coronary artery vessel 40 is shown withthe lesion partially closing the coronary vessel artery. In FIG. 10, theballoon 42 has been expanded so as to expand stent 44 to press againstthe vessel wall 46 and open the vessel lumen 48. The coating 50 on thestent 44 can now release the active agent(s) directly into the vesselwall to inhibit vascular smooth muscle proliferation.

The stent is introduced into the appropriate position as previouslydescribed for directing the balloon for angioplasty. However, in thiscase, the balloon is surrounded by the stent. As indicated above, whenthe balloon and stent are appropriately positioned, the balloon isexpanded expanding the vessel and the stent, and the stent assumes itsexpanded position and is held in place. By using a porous stent, theballoon can also provide the agent(s) as previously described. Afteradministration of the agent(s) from the balloon, the balloon is deflatedand retracted, leaving the stent in position to maintain the release ofthe agents, preferably a therapeutic mixture of L-arginine or itsbiologic equivalent and an agent which enhances a NO synthase agonist orstimulant, as described more fully hereunder.

Even more particularly, the presently disclosed “mixtures” may bedescribed in terms of their relative concentrations (grams) administeredas part of a continuous intracoronary, intra-arterial, intra-luminal,intramural, intravenous and intrapericardial infusions. In oneparticular embodiment, the formulation is administered as mixtures ofenhancers of no production (e.g., NOS agonist or HmgCoA reductaseinhibitors) with L-arginine encased in liposomes so as to providemaximum retention time of the mixture in any given vascular bed beingperfused by a catheter delivering the growth factor/L-arginineangiogenic mixture. In some cases the liposomes containing the mixturemay also contain genetic material for transfection of the geneticmaterial into the surrounding tissue of the vascular bed, In some casespellets containing the aforementioned mixtures may be directly implantedinto the myocardium at the time of coronary bypass graft surgery. In yetanother case, a therapeutic mixture of L-arginine and an angiogenicgrowth factor are repeatedly infused into the pericardial space via anindwelling infusion catheter.

Compositions of the present invention may be in the form of an agent(s)in combination with at least one other agent, such as stabilizingcompound, which may be administered in any sterile, bio-compatiblepharmaceutical carrier, including, but not limited to, saline, bufferedsaline, dextrose, and water. The compositions may be administered to apatient alone, or in combination with other agents, drugs or hormones.Pharmaceutically-acceptable carriers may also be comprised of excipientsand auxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of Remington's Pharmaceutical Sciences (Maack Publishing Co.,Easton, Pa.) hereby incorporated herein by reference in its entirety.The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. Such labeling would include amount, frequency, and method ofadministration.

Treatment with L-arginine or with other agents that increase eNOSactivity and NO production have been found to protect against vascularinjury in various experimental models. Since these treatments alsostimulate tPA (tissue plasminogen activator) production and/or inhibitproduction of PAI-1, it is likely that their protective effects are dueat least in part to effects in increasing tPA activity. Recently, manyagents whose principal actions are unrelated to eNOS activity have beenshown to have independent auxiliary actions through eNOS activation andNO production. These include organic nitrates, converting enzymeinhibitors, amrinone, nevilolol, S-nitroso-TPA, pravastatin andamlodipine. Doxazosin appears to have a similar auxiliary mechanism andthat it increases TPA levels as a result of effects on eNOS activationin ECs.

In an alternative embodiment of the invention, therapeutically effectiveamounts of a precursor of EDNO and these agents which have auxiliaryaction through eNOS (e.g., DOX) are combined prior to administration toa patient.

Doxazosin (DOX), an effective antihypertensive agent andα-adrenoreceptor antagonist, has been found to increase serum levels oftissue plasminogen activator (tPA). In fact, a wide variety ofvasoactive agents (e.g., bradykinin, muscarinic agonists and growthfactors) which increase tPA levels are also agonists of nitric oxidesynthase (NOS). DOX activity as a NOS agonist was investigated incultured bovine aortic EC using two methods to assess NO production:conversion of oxyhemoglobin to methamoglobin and a NO sensitiveelectrode. We found that DOX (10⁻⁷-10⁻⁵ M) produced a dose-relatedincrease (64-145%) in NO production. This increase in NO to DOX (10⁻⁶ M)was blocked by 72% by prior administration of the NOS inhibitor, L-NAME(5×10⁻⁴ M). In addition, the NO responses were accentuated by thepresence of supplemental L-arginine (5×10⁻⁴ M) by 65%. Acetylcholinealso produced a dose-related increase in NO production. This increase inNO to ACH (10⁻⁶ M) was blocked by 86% by prior administration of L-NAME.In addition, the NO responses were accentuated by 72% in the presence ofsupplemental L-arginine. DOX, therefore, appears to be a NOS agonist inEC.

For all experiments described herein, human coronary artery endothelialcells (passage 3-5-Clonetics) were maintained at 37°, 95% O₂ and 5% CO₂in Medium 199 (M199) supplemented with 5% fetal bovine serum (FBS,Hyclone), 10% iron supplemented FBS (Hyclone), thymidine (100 mg mL⁻¹),penicillin G (100 U mL⁻¹) and streptomycin (100 μmL⁻¹). In allexperiments below, the effects of pretreatment with L-NAME (10⁻³ M) inblocking the actions of DOX, and excess L-arginine (10⁻³ M) in reversingany L-NAME effect was examined. Acetylcholine, an eNOS agonist, was usedin all experiments as the positive control at concentrations of 10⁻⁷ and10⁻⁶ M.

Nitric Oxide Measurements. Methemoglobin—The effect of DOX on NOproduction in EC was determined using a photometric assay for conversionof oxyhemoglobin to methemoglobin. For this assay, EC grown toconfluency on microcarrier beads are placed into a water-jacketedchromatography column and superfused with a Kreb's-Ringer buffercontaining 3 μM oxyhemoglobin and 50 μM LA (L-arginine). Perfusate isthen directed into a flow-through cuvette in a dual wavelengthspectrophotometer and change in absorbency (415/405 nm) is measured.Experimental stimulation was carried out by 3 min infusion periods ofDOX added to buffer perfusion to yield final concentrations of 10⁻⁷ and10⁻⁶ M. For analysis, we determined the area under the curve for thechange in absorbency response/min caused by DOX assuming a one to onecorrespondence for NO and metHb production, the known stoichiometricbalance for this reaction. NO production was measured with a NO meterconnected to a polargraphic NO electrode as previously described. The NOsensor probe will be inserted vertically into 24-well plates containingconfluent EC such that the tip of the electrode is submerged 2 mm underthe surface of medium (above −1 ml). The reaction was initiated whendesired concentrations of DOX are added to the well. Calibrations wereperformed with S-nitroso-acetyl-penicillamine.

TPA assay. In the TPA assay, EC was grown to confluency in 24-wellplates and on experimental days, the medium was discarded and replacedwith 0.5 ml serum-free M199 containing 1% BSA and 50 μM LA and incubatedat 37% for 48 hrs in the presence of DOX (10³¹ ⁷ to 10⁻⁵ M) oracetylcholine (10⁻⁷ and 10⁻⁶ M) with and without L-NAME and excess LA.After incubation, the medium was harvested for determination of tPAcontent by an ELISA kit.

It would appear that DOX, like nitroglycerin, substance P andbradykinin, acts as a NOS agonist. It appears that the responses to DOXcan be magnified significantly with L-arginine supplementation. Itappears the overall therapeutic result with DOX is augmented to theextent they act as agonists of NOS. The fact that DOX is an agonist or astimulator of nitric oxide synthase has important implications. MixingDOX “in vitro” or “in vivo” with L-arginine may have an unforeseenbeneficial effect that is associated with excess L-arginine providingadditional substrate for NOS and the NOS being catalyzed toenzymatically increase the bio-transformation of L-arginine into nitricoxide (EDRF or EDNO) which would in turn amplify the overall therapeuticeffect.

Stimulation of NOS by DOX in the presence of excess L-arginine or othersubstrate precursor of native NO may be used to prevent, treat, arrest,or ameliorate any disease or condition which is positively affected byNO production. Such conditions include hypertensivecardiocerebrorenovascular diseases and their symptoms as well asnon-hypertensive cardiocerebrorenovascular diseases. The mixture isparticularly useful for subjects in need of native NO production fortherapeutic angiogenesis.

The ratio of DOX to L-arginine is preferably within the range 1:2 to1:50, Wt/Wt. For example, DOX/L-arginine at a ratio of 1:2 would include40 mg/day VEGF with 80 mg/day L-arginine. Where the ratio ofDOX/L-arginine is at a ratio of 1:20, for example, 20 mg/day DOX wouldbe administered with 400 mg/day L-arginine. The amounts above have beenfound to be effective, however, each route of administration (ie., IV,oral, transdermal, intracoronary, intra-arterial, etc.) may vary intheir requirements.

As discussed herein with regard to the other mixtures, theDOX/L-arginine mixture may be encased in liposomes so as to providemaximum retention time of the mixture in any given vascular bed beingperfused by a catheter delivering the DOX/L-arginine mixture. In somecases the liposomes containing the mixture of DOX and L-arginine mayalso contain genetic material which will code for the synthesis of thegrowth factor following transfection of the genetic material into thesurrounding tissue of the vascular bed. In some cases pellets containingthe aforementioned mixtures may be directly implanted into themyocardium at the time of coronary bypass graft surgery. In yet anothercase, a therapeutic mixture of L-arginine and DOX may be repeatedlyinfused into the pericardial space via an indwelling infusion catheter.

The therapeutically effective dose of DOX can be estimated initiallyeither in cell culture assays, e.g., of neoplastic cells, or in animalmodels, usually mice, rabbits, dogs, or pigs. The animal model may alsobe used to determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy.

An alternative embodiment of the present invention is based on a thefact that when cellular supply of L-arginine is limited, NOS utilizesmolecular oxygen as a lone substrate producing superoxide anion (O₂*—)and other reactive free radicals which can lead to cardiovasculardysfunction and the pathogenesis of disease.

The total intracellular concentration of L-arginine (0.1-1 mM) inendothelial cells (EC) greatly exceeds the K_(m) of eNOS for L-arginine(−3 μM). This suggests that eNOS is saturated with substrate and thatlevels of intracellular L-arginine are not limiting for NO production.However, other studies have shown that availability of L-arginine variesgreatly within the EC due to intracellular compartmentalization anddequestration in addition to degradation by arginase or the presence ofendogenous inhibitors of eNOS (i.e., asymmetrical dimethylarginine).Recently, it has also been shown that concurrent cellular L-argininetransport may be more important than intracellular L-arginine levels inproviding L-arginine to NOS for NO production. Therefore, totalintracellular concentration of L-arginine may not truly reflect theL-arginine available at the site of NOS action.

Supply of L-arginine may become limiting and reduce formation of NO innormal and pathological states. Treatment of guinea pigs with L-argininehas been shown to increase the duration of the vasodilatory response toacetylcholine under normal physiological conditions; prior stress withnorepinephrine infusion accentuates this enhancement process. It hasbeen demonstrated that acetylcholine and a Ca⁺⁺-ionophore which releaseNO can induce tolerance in isolated arterial rings. Tolerance wasassociated with depletion of L-arginine and was reversed with L-argininerepletion. L-arginine may also become limiting under pathologicconditions. Endothelial dysfunction in cardiomyopathic hamsters can bereversed by L-arginine. In addition, humans with acute hyperglycemiaexhibit intense vasoconstriction and impaired endothelial function whichcan be completely reversed by intravenous infusions of lowconcentrations of LA. Other diseases in which pathology is attributed toa deficiency of L-arginine include hypertension, atherosclerosis,restenosis—post coronary angioplasty and reperfusion injury. Similarly,addition of L-arginine in these circumstances also ameliorates thedeficit in endothelium-dependent relaxation.

Intracellular L-arginine is derived from several sources including thetransport of L-arginine into cells, amount of intracellular L-citrullinerecycled back to LA, rate of degradation of L-arginine (arginase),incorporation of L-arginine into proteins (compartmentalization) and theamount of L-arginine utilized upon activation of intracellular NOS.Uptake of L-arginine into EC occurs through two carrier-mediatedtransporters and passive diffusion. The saturable carrier-mediatedtransporters include a sodium-dependent active transporter, systemB^(α+) and a sodium-dependent transporter, system y⁺. The majority (80%)of L-arginine delivered into most cells is through the y⁺ transporter.Regulation of L-arginine transport appears to involve cellular membranepotential. Exposure of endothelial cells to hyperpolarizing agentsincluding ATP and bradykinin increases L-arginine uptake while adecrease in L-arginine transport was observed when cells were treatedwith agents that cause cellular depolarization. In addition, factorsthat reduce the activity of the y⁺ transporter, including free radicals,may also reduce L-arginine available for NOS.

When the balance of transporter regulatory factors is negative,L-arginine supply becomes limiting and subsequent production of O₂*—maycontribute to vascular and organ pathology. We compared the effects ofNOS agonists and NO donors on L-arginine uptake by EC. Effects of NOSstimulation on superoxide anion production were also assessed in thepresence and absence of L-arginine and the NOS antagonist, L-NAME.

FIG. 10 is a schematic representation of the hypothesized dynamics ofL-arginine supply to NOS. L-arginine levels are maintained primarilythrough the activity of the carrier-mediated Nα+-independenttransporter, y⁺, while the Na⁺-dependent transporter, B^(α+), andpassive diffusion account for less than 15%. Concurrent transport ofL-arginine to NOS may control NO production. However, L-arginine supplyto NOS can be limiting due to compartmentalization within EC, arginaseactivity or utilization of L-arginine by NOS. We believe that NO andsuperoxide anion reduce the activity of the y⁺ transporter and alsoreduce L-arginine available for NOS. Collectively, summation of supplyverses demand or availability of L-arginine to NOS will determinewhether NO or superoxide anion are formed.

Cellular Transport of L-arginine into BAEC. As can be seen in FIG. 11,initial data demonstrated that transport of cellular [³H]-LA into BAECoccurs linearly with time for up to 1 hour. FIG. 11 indicates the effectof B^(α+) and y⁺ transporters on cellular uptake of [³H]-L-arginine.Bovine aortic endothelial cells were incubated with uptake buffercontaining tritiated L-arginine and the amount of [³H]-L-argininedelivered to cells over time was determined as described in “Methods.”Dashed line, uptake of [³H]-LA by both B^(α+) and y⁺ transporters; solidline, uptake of [³H]-LA by y⁺ transporter. Data are presented as mean±S.E.M. We also found that the primary transporter of L-arginine intothese BAEC is the y⁺ transporter which was responsible for ˜85% of the[³H]-LA delivered to cells. The B^(α+) transporter system accounted foran average of 10% total transport. Passive diffusion as a percent oftotal cellular L-arginine uptake was variable and decreased as theperiod of uptake was increased, accounting for 5 to 1.5% of totalcellular L-arginine transport during 15 to 60 minutes of uptake,respectively.

Effect of NOS agonists on cellular uptake of LA. As can be seen in FIGS.12, 13, and 14, L-arginine transporter activity was augmented afteracute exposure to select NOS agonists. FIG. 12 indicates the effect ofbradykinin (BK, 1αM) on y⁺ transport of [³H]-LA in bovine aorticendothelial cells. Cells were exposed to BK and incubated withsodium-free uptake buffer containing tritiated L-arginine and the amountof [³H]-LA delivered to cells was determined as described in “Methods.”Data are presented as mean S.E.M; p<0.05 from control values. As can beseen in FIG. 12, bradykinin (BK) enhanced cellular transport ofL-arginine with maximum increase of 42% observed after 15 minuteexposure and slightly less but still marked increases of 39 and 16%occurring after treatment for 30 and 60 minutes, respectively. Prolongedexposure of BAEC to BK enhanced cellular uptake of L-arginine by 38%after 2 hour exposure. A similar magnitude of increase was also observedafter 3 and 5 hour exposure, with increases in transport of 19 and 22%,respectively.

FIG. 13 indicates the effect substance P (SP, 1 μM) on y⁺ transport of[³H]-LA in bovine aortic endothelial cells. Cells were exposed to SP andincubated with sodium-free uptake buffer containing tritiated L-arginineand the amount of [³H]-LA delivered to cells was determined as describedin “Methods.” Data are presented as mean S.E.M; p<0.05 from controlvalues. As can be seen in FIG. 13, substance P (SP) was also effectivein augmenting cellular uptake of L-arginine into cells. SP increased y⁺transport of L-arginine into cells by 24% after only 15-minutesexposure. This elevated L-arginine uptake was maintained for exposuresof 30 and 60 minutes with 24 and 21% increases, respectively. Inaddition, the effect of SP on cellular transport of [³H]-LA was enhancedafter pre-treatment with SP for more prolonged durations. After 2 hourexposure of BAEC to SP, y⁺ transporter activity was enhanced as much as34% from control values. This increase in transporter activity was alsomaintained after 3 and 5 hour exposure with cellular L-arginineincreases of 27 and 21%, respectively.

Effects of a third NOS agonist, acetylcholine (Ach) on the cellularuptake of [³H]-LA are shown in FIG. 14. FIG. 14 indicates the effect ofacetylcholine (Ach, 5 μM) on y⁺ transport of [³H]-LA in bovine aorticendothelial cells. Cells were exposed to Ach and incubated withsodium-free uptake buffer containing tritiated L-arginine and the amountof [³H]-LA delivered to cells was determined as described in “Methods.”Data are presented as mean S.E.M; p<0.05 from control values. Incubationwith Ach increased L-arginine transport over all time periods. A 22%increase of [³H]-LA uptake was observed after 2 minute exposure to Ach.After 15 minute addition of Ach, L-arginine uptake reached to a maximumincrease of 27%. Treatment with Ach for 30 or 60 minutes resulted in 16and 15.5% increases of L-arginine uptake, respectively.

Effect of NO donors on cellular uptake of LA. FIG. 15 indicates theeffect of s-nitroso-acetyl-penicillamin (SNAP, 200 gM; equivalent to 0.4μM NO) on y⁺ transport of [³H]-LA in bovine aortic endothelial cells.Cells were exposed to SNAP and incubated with sodium-free uptake buffercontaining tritiated L-arginine and the amount of [³H]-LA delivered tocells was determined as described in “Methods.” Data are presented asmean S.E.M; p<0.05 from control values. As can be seen in FIG. 15,treatment of endothelial cells with 200 μM SNAP (0.4 μM NO) markedlyincreased activity of the Y⁺ transporter by 37% occurring after tenminutes of exposure. This elevation was not seen after 30 minuteexposure. By 1 hour, uptake of [³H]-LA was reduced by 22%. Inhibitionwas maintained with 46, 45, and 36% reductions observed after 2, 3 and 5hour exposures to NO, respectively.

In order to confirm whether the reduction in cellular uptake of [³H]-LAwas due to NO released from SNAP, experiments were performed usinganother NO donor, DPTA-NONOate. Unlike SNAP, which donates large amountsof NO over a short time period (t_(1/2)˜10 minutes), the use ofDPTA-NONOate allows for a slower (t_(1/2)˜5 hours), more sustainedrelease of NO that is constant over time. FIG. 16 indicates the effectof dipropylenetriamine NONOate (D PTA, 10-0.01 μM; equivalent to 20-0.02μM NO) on y⁺ transport of [³H]-LA in bovine aortic endothelial cells.Cells were exposed to DPTA and incubated with sodium-free uptake buffercontaining tritiated L-arginine and the amount of [³H]-LA delivered tocells was determined as described in “Methods.” Data are presented asmean S.E.M; p<0.05 from control values. Exposure to 1 μM DPTA-NONOate (2μM NO) had no significant effect on y⁺ system at the earlier periods (15and 30 min); however significant inhibitions of 22, 24 and 29% forL-arginine transport were observed after 1, 2 and 4 hour exposures,respectively (data not shown). From FIG. 16, this repression appeared tobe concentration dependent with maximum inhibition of 20, 24 and 44%occurring after 2 hour exposure with concentrations of 0.01, 1, and 10μM (20, 2 and 0,02 μM NO), respectively.

Cellular superoxide anion formation—Effect of NOS agonists on cellularsuperoxide anion formation. In order to determine the effects ofextracellular L-arginine or NOS antagonist L-NAME on BAEC superoxideanion formation, experiments were performed in which cellular productionof superoxide anion was monitored alone (basal) and during treatmentwith SP (1 μM) or the calcium ionophore A-23187 (1 μM), with or withoutconcurrent presence of L-arginine or L-NAME supplementation. FIG. 17indicates the effect of L-arginine (LA, 5×10⁻⁴M) andn-ω-nitro-L-arginine methyl ester (L-NAME, 5×10⁻⁴M) on substance P (SP,1 μM) or calcium ionaphore, A-23187 (CI, 1 μM) induced superoxide anion(O₂*—) formation in bovine aortic endothelial cells (BAEC). BAEC weretreated with SP or A-23187 in the presence or absence of L-arginine orL-NAME and O₂*—production was determined over a 60 minute time periodand compared to basal levels as described in “Methods.” Data arepresented as mean S.E.M; p<0.05 from control values. FIG. 9 demonstratesthat O₂*—is produced by BAEC and that supplementation with L-NAME, butnot LA, prevented basal production of O₂*—by 100%. Addition of SP orA-23187 significantly increased O₂*—production above basal levels by 3.5and 2.5 fold, respectively. Concurrent treatment with either L-arginine(5×10⁻⁴M) or L-NAME (5×10⁻⁴M) effectively reduced O₂*—induced by SP by51 and 81%, respectively. Similar inhibitory effects of L-arginine andL-NAME on O₂*—production were observed when the calcium ionophoreA-23187 was used to induce NOS activation, with 60 and 58% inhibitionobserved with L-arginine and L-NAME, respectively.

The transport of L-arginine to cells is critical for maintainingadequate L-arginine levels such that optimal coupling of L-arginine withendothelial NOS (eNOS) can occur. Therefore, factors affecting the y⁺transporter system have the potential to limit the production of NO.Without ample LA, eNOS will solely utilize O₂ to form O₂*—that maycontribute to the pathogenesis of disease. As a consequence, controllingL-arginine supply and other factors affecting superoxide productionwould be beneficial in normal as well as pathological circumstances.

The cellular L-arginine transport system in BAEC is characterized here.The data presented herein confirms that the primary source of L-argininesupply is through activity of the system y⁺ transporter and thatdelivery of L-arginine into cells occurs linearly over two hours. Inaddition, we have verified that system B^(α+) transport activity andpassive diffusion contribute minimally to the delivery of L-arginineinto BAEC under basal conditions. Our experimental results were similarto those observed using human umbilical EC and porcine aortic EC. Theseexperiments were important to perform in order to determine whichtransport mechanism should be studied.

The data presented herein demonstrates BK causes an increase in cellularuptake of LA. These results are consistent with a study by Bogle et al.in which porcine aortic endothelial cells grown on microcarrier beadsincreased their cellular uptake of [³H]-LA in the presence of BK within10 minutes. In addition to these findings, we were able to demonstratethat this enhancement of cellular uptake of L-arginine was maintainedfrom 15 minutes through 2 hours exposure to BK. More importantly, wewere able to demonstrate an increase in y⁺ transporter activity for twoother NOS agonists, SP and Ach. As stated earlier, a negative change incellular membrane potential is thought to be the mechanism by which y⁺system activity is maintained. Hyperpolarization associated withstimulation of y⁺ system is thought to occur by first increasingintracellular Ca⁺⁺. This increase in Ca⁺⁺-dependent potassium channels(K_(ca)) resulting in K+ efflux and hyperpolarization. Since BK, SP andAch have also been shown to induce cellular hyperpolarization, thesedata suggest the increase in y⁺ transporter activity observed occurredby a similar mechanism.

Interestingly, our data for the NO donor, SNAP, depicts initialstimulation of the y⁺ transporter within 10 minutes followed by nochange and then inhibition of cellular L-arginine uptake with moreprolonged exposures to NO, a “cross-over” effect. An initial increase ofcellular uptake of L-arginine is expected as NO is known to causecellular hyperpolarization. However, longer exposures of 1 to 4 hoursresulted in a marked reduction of L-arginine transport. These data wereconfirmed by using a different NO donor, DPTA, to stimulate prolongedexposure of cells to NO. DPTA releases NO slowly over time and,therefore, was used to repeat the longer durations of NO exposure.Although one might expect to see a continued increase of y⁺ transporteractivity with NO exposure similar to that observed using NOS agonists,there is evidence that oxidative properties of NO may be responsible forthe reduction of cellular L-arginine transport seen with longer exposureperiods. It has been demonstrated that NO, through constant gas infusionand release from SNAP, decreases y⁺ system transporter activity. Thenegative effect of NO on y⁺ transport of L-arginine into cells wasdetermined to be associated with oxidation of sulfhydryl moieties in thetransporter proteins since treatment with disulfide reducing agentdithiothreitol restored transporter activity. Furthermore, treatment ofendothelial cells with sulfhydryl reactive chemicals N-ethylmaleimide(NEM) and acrolein reduced y⁺ transporter activity. Collectively, thesedata suggest that the effects of NO on cellular y⁺ L-arginine transportactivity are two-fold. The initial effect seen upon acute exposure ismore likely due to the hyperpolarizing properties of NO while the latterinhibitory effects observed with more prolonged exposure to NO may bethe result of a summation of cell hyperpolarizing and transportoxidizing properties of NO, the latter becoming more predominant.

The biphasic effect in transport function over time noted for SNAP wasnot observed in cells treated with prolonged exposure to NOS agonists.It would be expected that stimulation of NOS would also increase NOproduction and oxidation of the y⁺ transporter system resulting ininhibition of L-arginine uptake similar to that observed with SNAP. Oneexplanation for lack of biphasic action with NOS agonists could be thatthe amount of NO produced upon NOS activation is far less than theamount of NO released from SNAP. Therefore, levels of NOS derived NOnever accumulate high enough for significant oxidation of the y⁺transporter. Another possibility to explain the lack of inhibition ofL-arginine transport with NOS agonist is the fact that upon stimulationwith NOS, L-arginine is converted to the intermediateNG-hydroxyl-L-arginine (l-HOArg) prior to forming L-citrulline and NO.L-HOArg is known to be an antioxidant and an inhibitor of arginase.Therefore, the L-HOArg intermediate may provide protection fromoxidation by newly formed NO. By preventing the metabolism of L-argininein the ornithine cycle, the net amount of L-arginine available for eNOSmay increase and lead to a reduction in O₂*—formation. Both of theseactions should protect the system y⁺ transporter from inactivation.

Hence the transport of L-arginine into cells via y⁺ transport system maybe unfavorably altered with elevated levels of NO. High concentrationsof NO could occur during circumstances in which NOS is constantlystimulated. Pathophysiological conditions associated with increased NOSactivity include hypoxia, hyperglycemia and hypertensive states mediatedby elevations in angiotensin II (high renin essential and renovascularhypertension). The combination of increased NOS activity (L-argininedemand) and decreased arginine uptake (L-arginine supply) has thepotential to create an L-arginine deficiency (“demand-supply mismatch”)which can result in the increased superoxide anion production seen instates such as ischemia-reperfusion injury. Increased O₂*—production andNOS activity have also been shown to be associated with hyperglycemia.

The production of O₂*—in BAEC alone and during treatment with NOSagonists is characterized herein. In addition, the effects of basal andNOS agonist induced O₂*—production with concurrent addition ofL-arginine and L-NAME has been presented herein. The data demonstratethat BAEC produce O₂*—which increases with time and supplementation withL-NAME reduces basal O₂*—production. Since L-NAME is a selective NOSantagonist, this suggests that primary source of basal O₂*—observed isfrom eNOS. Stimulation of BAEC with SP or A-23187 produced amounts ofO₂*—much greater than basal levels. Interestingly, a striking reductionof O₂*—production was observed upon extracellular addition of eitherL-arginine or L-NAME, following treatment with SP and A-23187. Thesedata also suggest that excessive O₂*—formation associated with agonistinduced eNOS activation, but not basal production, can be amelioratedwith L-arginine supplementation.

Collectively, our findings strongly suggest that although intracellularL-arginine levels far exceed the concentration of L-arginine required byNOS for NO production, the amount of L-arginine available forutilization by NOS can be insufficient especially in conditions ofchronic eNOS stimulation. The explanation for this L-arginine paradoxmay be provided by the work of McDonald and colleagues. Using porcinepulmonary artery endothelial cells with antibodies specific forcaveolin, eNOS and the y⁺ transporter, McDonald et al. demonstrated thatall of these proteins are co-localized within the plasma membranecaveolae. This suggests that eNOS associated with this complex issequestered from overall intracellular L-arginine and relies on the denovo transport of L-arginine into the cell via the y⁺ transporter withinthe caveolae for NO production. If the transporter becomes damages asseen with oxidation, L-arginine supply could immediately become limitingand may be the basis for endothelial dysfunction. In addition, thiseNOS/y⁺ transporter-caveolae complex may explain why endothelialdysfunction is quickly reversed with increasing extracellular LA. Oncethe transporter is turned off, L-arginine concentration gradientincreases and delivery of L-arginine into cells is shifted towardspassive diffusion. Therefore, extracellular supplementation ofL-arginine may be helpful in driving passive diffusion of L-argininewhen the integrity of carrier-mediated transporters cinnot bemaintained.

We believe that concurrent L-arginine supply to NOS via system y⁺,independent of overall intracellular L-arginine, is critical inestablishing and maintaining vascular function. Factors including NOSagonists and NO itself appear to control y⁺ activity and the summationof these factors is critical in determining NO and superoxide anionformation, both of which contribute to vascular dysfunction and disease.

The subject methodology and devices provides an alternative treatment toand substantially reduce the occurrence of restenosis after vascularinjury. The methodology is simple, can be performed in conjunction withthe procedure resulting in the vascular injury, and is expected to bevery effective. With the present invention, a procedure which has beencommonly used can find expanded application as a result of the reducedincidence of restenosis.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

1. A composition comprising citrulline and an Hmg-CoA reductaseinhibitor.
 2. The composition of claim 1, wherein said citrulline isL-citrulline.
 3. The composition of claim 1, wherein said citrulline isa salt of L-citrulline.
 4. The composition of claim 1, wherein saidcitrulline is L-citrulline hydrochloride.
 5. The composition of claim 1,wherein the Hmg-CoA reductase inhibitor is pravastatin.
 6. Thecomposition of claim 1, wherein the Hmg-CoA reductase inhibitor isselected from the group consisting of atorvastatin, cerivastatin,simvastatin, lovastatin, compactin, fluvastatin, mevastatin,fluindostatin, velostatin and dalvastatin.
 7. The composition of claim1, further comprising a pharmaceutically acceptable carrier.
 8. Thecomposition of claim 1, wherein the composition is formulated for a formof administration selected from the group consisting of intravenous,buccal, intracoronary, intra-arterial, intrapericardial intramuscular,tropical, intranasal, rectal, sublingual, oral, subcutaneous, patch andinhalation.
 9. A therapeutic composition comprising a therapeuticallyeffective amount of citrulline, an Hmg-CoA reductase inhibitor and apharmaceutically acceptable carrier.
 10. The composition of claim 9,wherein said citrulline is L-citrulline.
 11. The composition of claim 9,wherein said citrulline is a salt of L-citrulline.
 12. The compositionof claim 9, wherein said citrulline is L-citrulline hydrochloride. 13.The composition of claim 9, wherein the Hmg-CoA reductase inhibitor ispravastatin.
 14. The composition of claim 9, wherein the Hmg-CoAreductase inhibitor is selected from the group consisting ofatorvastatin, cerivastatin, simvastatin, lovastatin, compactin,fluvastatin, mevastatin, fluindostatin, velostatin and dalvastatin. 15.The composition of claim 9, wherein the composition is formulated for aform of administration selected from the group consisting ofintravenous, buccal, intracoronary, intra-arterial, intrapericardial,intramuscular, tropical, intranasal, rectal, sublingual, oral,subcutaneous, patch and inhalation.
 16. A method of treating acardiovascular disease in a subject in need thereof comprisingadministering a composition comprising citrulline and an Hmg-CoAreductase inhibitor, wherein the level of nitric oxide in said subjectis increased.
 17. The method of claim 16, wherein the Hmg-CoA reductaseinhibitor enhances nitric oxide synthase activity.
 18. The method ofclaim 16, wherein said citrulline is L-citrulline.
 19. The method ofclaim 16, wherein said citrulline is a salt of L-citrulline.
 20. Themethod of claim 16, wherein said citrulline is L-citrullinehydrochloride.
 21. The method of claim 16, wherein the Hmg-CoA reductaseinhibitor is pravastatin.
 22. The method of claim 16, wherein theHmg-CoA reductase inhibitor is selected from the group consisting ofatorvastatin, cerivastatin, simvastatin, lovastatin, compactin,fluvastatin, mevastatin, fluindostatin, velostatin and dalvastatin. 23.The method of claim 16, wherein the composition further comprises apharmaceutically acceptable carrier.
 24. The method of claim 16 whereinsaid cardiovascular disease is selected from the group consisting ofhypertension, atherosclerosis, restenosis—post coronary angioplasty, andreperfusion injury.
 25. A method of stimulating nitric oxide synthase ina host in need thereof comprising administering citrulline and anagonist of nitric oxide synthase, said agonist being an Hmg-CoAreductase inhibitor.
 26. The method of claim 25, wherein said citrullineis in excess to said agonist.
 27. The method of claim 25, wherein atherapeutically effective amount of said citrulline is combined with atherapeutically effective amount of an Hmg-CoA reductase inhibitor priorto said administration.
 28. The method of claim 27, wherein the Hmg-CoAreductase inhibitor is pravastatin.
 29. The method of claim 27, whereinthe Hmg-CoA reductase inhibitor is selected from the group consisting ofatorvastatin, cerivastatin, simvastatin, lovastatin, compactin,fluvastatin, mevastatin, fluindostatin, velostatin and dalvastatin. 30.The method of claim 25, wherein said Hmg-CoA reductase inhibitorenhances nitric oxide production.